Breakthroughs in our understanding of circadian timekeeping in the brain will be necessary to ameliorate the metabolic and psychological pathologies that accompany human circadian misalignment. The goal of this proposal is to critically test and extend current models of clock neuron network modulation and entrainment in Drosophila. The nervous system of this fly has several remarkable features that make it a valuable model for circadian timekeeping. We have recently developed a new set of live-imaging tools for the interrogation of central neuron networks in the adult Drosophila brain. These methods have made it possible to measure Ca2+ and cAMP dynamics in central neuronal networks in concert with the acute stimulation of genetically defined neuronal subsets. Using these new techniques, coupled with well-established genetic, anatomical, and behavioral methods, we will investigate the neurophysiological basis of circadian modulation and entrainment, two closely related processes that are central to the pathologies associated with the circadian misalignment that accompanies modern life. In our first aim we will determine the roles that cholinergic and GABAergic modulation of critical ventral clock neurons plays in the entrainment of sleep/activity rhythms to light cycles. In our second aim, we will identify and physiologically characterize the neural light input pathways that modulate the circadian clock neuron network and genetically determine the roles these pathways play in light entrainment. In our third and final aim, we will employ new methods of circuit interrogation to determine the physiological nature of the modulatory connections between the ventral and dorsal clock neuron classes, how these connections are daily and seasonally adjusted, and will use genetic methods to identify the specific roles that two ventral-to-dorsal peptidergic connections play in the maintenance and adjustment of circadian timekeeping. The goal of this proposal is to advance our understanding of circadian timekeeping and entrainment in the brain. We believe that the fruits of this work will ultimately aid the development and implementation of interventions designed to alleviate the significantly adverse metabolic and psychological effects of circadian dysfunction. PUBLIC HEALTH RELEVANCE: The proper alignment of internal circadian clocks with the 24-hour rhythm of the environment is critical for human health and psychological well-being. New molecular imaging methods make it possible for the first time to examine the modulation of neuronal clocks within the brain of the fly, Drosophila melanogaster, an organism that continues to enrich and inform our understanding of circadian timekeeping in the brain. Here we propose experiments to understand how neuronal clocks are physiologically adjusted by internal and external cues, the results of which will ultimately aid in the development of interventions designed to alleviate the adverse metabolic and psychological effects of human circadian dysfunction.